QuantiNova LNA PCR Custom Assays

High-performance assays created with a convenient design tool and the same algorithm used for our predesigned assays

Custom assays to match your exact target and need, including detection of novel transcripts or a specific isoform or splice variant

Ability to submit up to 10 sequences and design assays to differentiate or detect all sequences

Short, LNA-enhanced primers with high specificity and greater flexibility in assay positioning

Robust and reproducible results in just 2 hours using the QuantiNova SYBR® Green PCR Kit

QuantiNova LNA PCR Custom Assays provide accurate and sensitive gene expression analysis of any RNA target using LNA-enhanced, SYBR® Green-based detection. Our robust design algorithm provides full Ensembl database support for human, mouse and rat targets and streamlines assay creation. Custom assays are well-suited for targets for which no predesigned assay is available, for discriminating splice variants and for verifying novel mRNAs or lncRNAs. Optimized using QuantiNova chemistry, the simple QuantiNova LNA PCR workflow can be completed in just 2 hours and is robust enough to support room-temperature setup, automation and delayed run of assays. Predesigned assays, pathway-focused panels and custom panels are also available and can be used together in the same experimental setup.

Need a quote for your research project or would you like to discuss your project with our specialist team? Just contact us!

The PCR performance of QuantiNova LNA PCR Assays was evaluated using a commercially available DNA template (gBlock). DNA templates for ATP6V0D2, BRCA2, CCND1, CDK6, and PPA1 were serially diluted from 1x109 to 10 copies and quantified with QuantiNova LNA PCR Assays using a Bio-Rad CFX96 or Bio-Rad CFX384 instrument. The QuantiNova LNA PCR Assays exhibited high linear range combined with outstanding sensitivity and near optimal efficiency. This high linearity along the entire input range is essential to prevent biases in calculations of relative expression.

Ten-fold dilutions of input cDNA ranging from 100 ng to 1 pg were prepared and quantified with QuantiNova LNA PCR Assays for 7 different mRNA targets on a Bio-Rad CFX96 or Bio-Rad CFX384 instrument. The assays exhibited accurate results at each dilution point demonstrating an efficiency of the assays over the full dilution range, as demonstrated by the example amplification curve shown (IFI16, 10 ng to 1 pg).

Performance

Highly sensitive and specific LNA-enhanced PCR

QuantiNova LNA PCR Custom Assays are created using stringent design criteria and lab-validated algorithms to deliver the highest real-time PCR specificity and efficiency and the most reliable and accurate gene expression analysis results.

The high binding affinity of LNA bases increases the flexibility of primer placement on the transcript, so we can use intelligent positioning to design assays for otherwise difficult-to-analyze targets. This gives you better target discrimination and robust and reliable quantification, even for AU-rich targets, low-abundance transcripts, targets with high secondary structure and highly complex samples.

Created using a sophisticated LNA design algorithm

Twenty years of LNA design experience has enabled us to develop and optimize our sophisticated LNA design algorithm, which incorporates over 50 different parameters – all thoroughly lab-validated against highly stringent performance criteria – to guarantee the most optimal assay for successful target detection. This proprietary algorithm has been used to design over 1.3 million assays and is available for your specialized requests through our convenient custom assay builder in GeneGlobe.

Principle

The custom design process

The QuantiNova LNA PCR Custom Assay design tool lets you easily design highly sensitive and specific, LNA-enhanced PCR assays for any mRNA or lncRNA not available as a predesigned assay. Using the advanced QuantiNova PCR Assay design algorithm, numerous assay combinations are evaluated based on more than 50 different criteria to find the optimal assay for your target within a few minutes. The tool has been designed for human, mouse and rat mRNA and lncRNA targets of at least 55 nucleotides and blasts against the species-relevant databases, including Ensembl and RefSeq. For known gene targets, assays are designed as intron-spanning by default, similar to the predesigned assays, to prevent the risk of gDNA amplification.

Uniquely advanced assay design options

The custom assay design tool includes options to create transcript-specific designs for discriminating splice variants, SNPs or isoforms. Alternatively, designs for assays common to all transcript variants can also be created. You can submit up to 10 different target sequences at once and design either ten different assays, or for related transcripts, you can design a common assay for all.

Robust and high-performance SYBR® Green -based detection

QuantiNova LNA PCR Assays use SYBR® Green-based detection, which, when used with a highly specific PCR system, well-designed primers and optimized reaction conditions, offers several advantages over hydrolysis probe detection. First, it is possible to perform melting curve analysis immediately following the PCR, so you can confirm high specificity of the reaction and rule out formation of primer–dimers or non-specific amplicons. It also provides higher flexibility in the assay design, because you only need two primers, instead of two primers and a probe. Furthermore, while the primers ensure the specificity of the detection, the dye’s binding properties enable analysis of many different targets without having to synthesize target-specific, labeled probes, reducing your assay setup and running costs.

Reference Gene Assays can be easily combined with the custom assays

A wide selection of human, mouse and rat Reference Gene Assays are available to enable high-quality data normalization and ensure reliable results. These assays target endogenous coding RNAs, long non-coding RNA and small nucleolar RNA molecules that are typically constitutively expressed in a wide variety of tissues. The Reference Gene Assays are FAM labeled and have been functionally validated as reference genes for the QuantiNova LNA PCR system and work optimally with the QuantiNova RT and PCR reagents.

Normalization of mRNA/lncRNA qPCR results

Normalization removes technical and biological inter-sample variation unrelated to the biological changes under investigation. Proper normalization is critical for correct analysis and interpretation of results from real-time PCR experiments. Most commonly, stably expressed reference genes are used for normalization.

It is generally recommended to test several endogenous control reference gene candidates before setting up your actual mRNA/lncRNA expression analysis. These candidates should be chosen from genes expected to be stably expressed over the whole range of samples under investigation. They could be stably expressed mRNAs or lncRNAs selected based on literature or preexisting data (e.g., NGS or qPCR panel screening). The QuantiNova LNA PCR system offers validated reference gene assays for RNAs that tend to be stably expressed and are therefore good candidates as reference genes.

All reference gene candidates should be empirically validated for each study. One option for normalizing PCR panel when profiling a large number of mRNAs/lncRNAs is to normalize against the global mean – the average of all expressed mRNAs/lncRNAs. This can be a good option in samples with a high call rate (expressed genes) but should be used with caution in samples with low call rates. It is also not a good option in samples for which the general gene expression level is changed. Further guidance on normalization can also be found in the GeneGlobe Data Analysis Center.

User-friendly data analysis

The complimentary, web-based data analysis tool in the GeneGlobe Data Analysis Center includes a user-friendly wizard to guide you step-by-step through the normalization and analysis of your data and generates publication-ready results figures.

Procedure

Trusted QuantiNova chemistry

QuantiNova LNA PCR Custom Assays are designed using the time-tested, high-performance QuantiNova chemistry, which offers you the choice between one- or two-step qRT-PCR. Best results are obtained when reverse transcription is performed with the QuantiNova Reverse Transcription Kit. The resulting cDNA is then quantified by qPCR using the master mixes of the QuantiNova SYBR® Green PCR Kit combined with your designed QuantiNova LNA PCR Custom Assay.

For one-step qRT-PCR, we recommend using the QuantiNova SYBR® Green RT-PCR Kit, which offers quick reaction setup without need for optimization of reaction conditions. Simply add primers and RNA template to the ready-to-use RT-PCR master mix and start the reaction.

Fast and easy workflow

The fast and easy workflow can be completed in just 2 hours with minimal hands-on time and can be automated to save even more time and labor. The simple protocol has been tested for compatibility on all major brand qPCR cyclers (see figure QuantiNova LNA PCR Assays deliver consistent performance across all thermal cycler instruments). Furthermore, the cDNA generated in the RT step can be used across the entire QuantiNova LNA PCR System, allowing seamless transition from assays to panels depending on your research needs, saving you time and sample.